1. Field of the Invention
This invention relates to the monitoring and screening of long-term blood glucose levels in patients afflicted with diabetes mellitus. In particular, this invention relates to a method of isolating fast hemoglobins from their glucose dependent Schiff base precursors and other hemoglobin components present in human blood.
It has been known for some time that the quantity of hemoglobin A.sub.1 (HbA.sub.1), a glycosylated form of adult hemoglobin (HbA), is higher in the blood of diabetic persons than in that of normal persons. Hemoglobin A.sub.1 itself consists of several components, of which the main ones have been identified as HbA.sub.1a, HbA.sub.1b and HbA.sub.1c. These three components are collectively known as the "fast hemoglobins," since they elute through a chromatographic column relatively quickly. The precursors to these components are labile adducts in which the linkage between the glucose molecule and the hemoglobin molecule in an aldimine linkage (hereinafter referred to as a "Schiff base"). Due to the high reaction rate involved in its formation from glucose and hemoglobin A as well as its high tendency to dissociate back to these starting materials, the Schiff base level reflects short-term fluctuations in the blood glucose levels, rather than the long-term levels sought to be determined in a meaningful diabetic analysis. For this reason, analyses without Schiff base removal are often poor indications of a patient's ability to regulate glucose.
It is therefore desirable to find a method for determining the hemoglobin A.sub.1 content of human blood without interference from Schiff base precursors.
2. Description of the Prior Art
A general discussion of glycosylated hemoglobins and their relevance to diabetes mellitus is offered by Bunn, et al., Science, 200, pp. 21-27 (1978). The use of ion exchange resins is described by Chou, et al., Clin. Chem., 24(10), pp. 1708-1710 (1978) and in a series of U.S. patents to Acuff: Nos. 4,142,855, 4,142,856, 4,142,857 and 4,142,858 (all issued on Mar. 6, 1978), 4,168,147 (issued on Sept. 18, 1979) and 4,238,196 (issued on Dec. 9, 1980).
Known methods for removing Schiff base adducts include saline incubation of erythrocytes and dialysis of the hemolysate. The former is described by Goldstein, et al., Diabetes, 29, pp. 623-628 (1980), Svendsen, et al., Diabetologia, 19, pp. 130-136 (1980) and Chou, et al., Clin. Chem., 24(10), pp. 1708-1710 (1978). The latter is described by Goldstein, et al., supra, and Widness, et al., J. Lab. Clin. Med., 95(3), pp. 386-394 (1980).
Accurate analysis for HbA.sub.1c without prior removal of Schiff base has been achieved by a colorimetric technique using acid hydrolysis followed by treatment with thiobarbituric acid. This is described in Svendsen, et al., supra.
The use of borate ion in conjunction with an anion exchanger is disclosed in Bunn, et al., Advances in Hemoglobin Analysis, pp. 83-94, Alan R. Liss, Inc., N.Y. (1981), to retard the elution of glycosylated moieties in a sample where no Schiff base is present.